The present invention relates to a process for regulating actin polymerization in T lymphocytes. The present invention also relates to assays and methods useful for identifying compounds that regulate actin polymerization in a T lymphocyte.
Mammalian cells have cytoskeletal networks that are associated with their plasma membrane. The cytoskeleton is comprised of a dense network of actin filaments and associated actin-binding proteins. Components of both the cytoskeletal network and the plasma membrane are important for cellular signalling by, for example, localizing and focusing critical signalling molecules.
Certain mammalian cells comprise multichain surface receptors that enable a cell to respond to changes in the environment outside of the cell. One such multichain receptor is a T cell receptor (TCR) located on the surface of T lymphocytes. A TCR is a multichain, heteromeric structure composed of an antigen binding domain comprising xcex1 and xcex2 chains, and non-covalently associated signal transducing complexes, including CD3-xcex3, xcex4 and xcex5 chains, and the xcex6 chains. Signal transduction events produced by TCR ligation with major histocompatibility complexes (MHC) induce a variety of cytoplasmic metabolic changes. For example, gene transcription and production of interleukin-2 (IL-2) are promoted by TCR ligation with MHC molecules.
Abnormalities in T lymphocyte function can arise through deregulation of signalling in T cells. Such diseases include, for example, autoimmune diseases, immunodeficiency diseases and immunoproliferative diseases. T lymphocyte function also contributes to graft rejection. To develop compounds that regulate the activity of molecules involved in T cell function, there must be an understanding of the molecules and interactions involved in such T cell related diseases.
Prior investigators have suggested that ligand binding converts surface immunoglobulin (Ig) to a detergent insoluble form, and that Ig receptors subsequently undergo extensive degradation accompanied by the appearance of a detergent soluble membrane product (Braun et al., J. Immunol. 128:1198-1204, 1982). Parsey et al. (J. Immunol. 151:1881-1893, 1993) hypothesize about a connection between actin polymerization and the ability of immobilized anti-CD3 antibodies to stimulate changes in cell shape and F-actin morphology. Furthermore, the expression of four src-family genes associated with T cell activation was shown to be specifically blocked by cyclosporine (Furue et al., J. Immunol. 144(2): 736-739, 1990). Prior investigators, however, have failed to teach or appreciate that actin polymerization in T lymphocytes is specifically regulated by the presence of a particular motif (i.e., an immunoreceptor tyrosine-based activation motif; ITAM) of a xcex6 chain or xcex5 chain of a TCR.
Although therapeutics exist that regulate immune activity in an animal, problems have arisen due to the non-specific nature and harmful side effects of such drugs. Despite a long-felt need to discover compounds that specifically regulate T cell activity with minimal side-effects, the complexity and lack of understanding of signal transduction networks in a T cell has hindered the development of such compounds. The present invention offers a method and product that permits regulation of specific steps of a signal transduction pathway in cells having ITAM-containing receptors.
Despite the complexity of signal transduction networks in cells, the present invention provides for a method to regulate actin polymerization in T lymphocytes by controlling the step in the signalling pathway in which an ITAM of the xcex6 or xcex5 chain of a TCR interacts with a tyrosine kinase and/or an adaptor molecule. The advantages arising from this invention include the specific regulation of a step in a T lymphocyte signalling pathway that can regulate T lymphocyte growth, differentiation, homing, proliferation and death. The present inventors are the first to appreciate the specific molecular interactions involved in the actin polymerization steps within a T lymphocyte, and thus, are the first to propose a method and product that targets this particularly important event within a T lymphocyte.
One aspect of the present invention includes a method to identify compounds capable of regulating actin polymerization in a T lymphocyte, comprising: (a) contacting a putative regulatory compound with a T lymphocyte having a T cell receptor chain selected from the group consisting of a zeta chain and an epsilon chain, to form a contacted lymphocyte; (b) combining the contacted lymphocyte with a molecule capable of inducing the phosphorylation of the zeta chain or the epsilon chain; and (c) assessing the ability of the putative regulatory compound to regulate actin polymerization in the lymphocyte. In particular, the method further comprising assessing the amount of interleukin-2 produced by the lymphocyte.
Another aspect of the present invention includes a method to regulate actin polymerization in a T lymphocyte, comprising contacting a T lymphocyte with an effective amount of a regulatory reagent that is capable of altering the activity of an immunoreceptor tyrosine-based activation motif (ITAM) of a xcex6 chain of a T cell receptor. A preferred ITAM to regulate using the present method comprises the amino acid sequence SEQ ID NO:1. Regulation of actin polymerization by the present method preferably alters a T lymphocyte function including growth, differentiation, homing, proliferation, apoptosis and anergy.
The present invention also includes a method to regulate actin polymerization in a T lymphocyte, comprising contacting a T lymphocyte with an effective amount of a regulatory reagent that alters the activity of an immunoreceptor tyrosine-based activation motif of an xcex5 chain of a T cell receptor. A preferred ITAM to regulate using the present method comprises the amino acid sequence SEQ ID NO:2.
One embodiment of the present invention includes a cellular system where a src-family tyrosine kinase is contacted with a T cell receptor chain selected from the group consisting of a xcex6 chain and an xcex5 chain that is regulated by said src-family tyrosine kinase, the improvement comprising regulating actin polymerization by contacting a T lymphocyte with a reagent capable of binding to a protein including a third ITAM of a xcex6 chain, an ITAM of an xcex5 chain and an SH2 domain.
Another embodiment of the present invention includes a formulation capable of regulating actin polymerization in a T lymphocyte, the formulation comprising: (a) a regulatory reagent that alters the activity of a molecule including an immunoreceptor tyrosine-based activation motif of a xcex6 chain of a T cell receptor and an immunoreceptor tyrosine-based activation motif of a xcex5 chain of a T cell receptor in a cell; and (b) a pharmaceutically acceptable carrier.
The present invention also includes a kit to identify compounds capable of regulating actin polymerization in a T lymphocyte, the kit comprising: (a) a cell comprising a T cell receptor chain selected from the group consisting of a xcex6 chain, an xcex5 chain, and actin monomers; and (b) a means for detecting the polymerization of the actin monomers.